Dried blood spots (DBSs) are an economical and convenient alternative to serum/plasma, which allow for the serological and molecular study of different pathogens. Sixty-four blood samples were collected by venipuncture and spotted onto Whatman¿ 903 cards to evaluate the utility of DBSs and the effect of the storage temperature for 120 days after sample collection to carry out serological diagnosis. Mumps, measles and rubella IgG were investigated from DBSs and plasma using an automated chemiluminescent immunoassay. Using a calculated optimal cut-off value, the serological evaluation of mumps, measles and rubella using DBSs achieved high sensitivity (100%, 100% and 82.5%, respectively) and specificity (100%, 87.5% and 100%, respectively). The correlation observed between the plasma and the DBSs processed after sample collection was high (0.914¿0.953) for all antibodies studied, both considering hematocrit before sample elution or not. For the different storage conditions, the correlation with plasma was high at 4 °C (0.889¿0.925) and at ¿20 °C (0.878¿0.951) but lower at room temperature (0.762¿0.872). Measles IgG results were more affected than other markers when DBSs were stored at any temperature for 120 days. To summarize, hematocrit does not affect the processing of DBSs in the study of serological markers of mumps, measles and rubella. DBS stability for serological diagnosis of mumps and rubella is adequate when samples are stored at ¿20 °C or 4 °C, but not at room temperature, for a period of 4 months.